Floral organ-specific gene and its promoter sequence

ABSTRACT

The flower organ-specific promoter is contained in the sequence consisting of nucleotides of positions from 1 to 1234 in the nucleotide sequence represented by SEQ ID NO:3. The promoter includes sequences derived from the above sequence by deletion, substitution, insertion or addition of one or more nucleotides and having a flower organ-specific promoter activity and also those obtainable by using the nucleotide sequence represented by SEQ ID NO:1 as a probe and having a flower organ-specific promoter activity.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/JP97/04892 which has an Internationalfiling date of Dec. 26, 1997 which designated the United States ofAmerica, the entire contents of which are hereby incorporated byreference.

TECHNICAL FIELD TO WHICH THE INVENTION BELONGS

This invention relates to a gene showing specific expression inmonocotyledon flower organs and its promoter sequence. This inventionfurther relates to a chitinase acting as a defensive mechanism againstpathogenic bacteria and a chitinase gene.

PRIOR ART

There have been reported some cases of the isolation of genes which areexpressed specifically in flower organ, for example, anther-specificgenes and pistil-specific ones. However, only a few genes specific toanother have been reported as genes which are isolated frommonocotyledons and for which the promoter sequences have been clarified.

These reports are exemplified by JP (Kohyo) HEI 6-504910, Tsuchiya etal. Plant Mol. Biol. 26, 1737-1746, 1994, etc. in which the nucleotidesequences of rice anther-specific genes, their expression profiles, etc.are indicated.

Promoters exhibiting expression specifically in flower organ arerequired in order to artificially improve the morphology of plant flowerorgans, in particular germ organs, or physiological phenomena or toanalyze functions of various genes in flower organs. In monocotyledonswhich represent major cereals, however, few genes expressed exclusivelyin flower organs have been isolated hitherto. In particular, there hasbeen reported no promoter sequence showing predominant expression inpistil which is the female germ organ or lodicule which regulatesflowering.

Although chitinase (EC 3.2.1.14), which seemingly acts as a defensivemechanism against pathogenic bacteria and fungi, can be cited as anexample of genes expressed in flower organ, most of the chitinases ofplant origin reported so far are constitutively expressed not only inflower organs but also in roots (see, for example, Neale et al. ThePlant Cell, 2, 673-684, 1990). Exceptionally, chitinases such as potatoSK2 (Wemmer et al. Planta 194, 264-273, 1994) and tomato Chi2;1(Harikrishna et al. Plant Molecular Biology, 30, 899-911, 1996) showstyle-specific expression.

On the other hand, there have been isolated some chitinases ofmonocotyledons. For example, Zhu ant Lamb (Mol. Gen. Genet., 226,289-2961991) isolated a chitinase called RCH10 from rice and reportedthat the gene of this enzyme was constitutively expressed in root underaseptic conditions. Further, Zhu et al. (BIO/TECHNOLOGY, 12, 807-812,1994) constructed tobacco with enhanced tolerance to pathogenic bacteriaby using the above-mentioned gene together with an alfalfa glucanasegene.

There has been no report in monocotyledons, however, about a chitinasewhich is not expressed at a detectable level in root, being expressedexclusively in flower organs.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel flowerorgan-specific promoter sequence enabling genetic manipulations ofpistil or lodicule which were impossible in the prior art particularlyin monocotyledons.

Another object of the present invention is to provide a novel chitinasewhich makes it possible to impart to plants a general resistance againstpathogenic bacteria and fungi containing chitin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 consists of photographs showing the results of Northern analysison RPC175 gene.

FIG. 2 consists of photographs showing the results of RT-PCR analysis onRPC175 gene.

FIG. 3 consists of a photograph showing the results of genomic Southernanalysis on RPC175 gene.

FIG. 4 consists of photographs showing the results of an experiment fordetermining the transcription initiation point of RPC175 gene by theprimer extension method (SEQ ID NO:5).

FIG. 5 is a model view showing the structure in the 3′ side of thepromoter of RPC175 gene (SEQ ID NO:6).

FIG. 6 is a drawing illustrating a comparison of the restriction maps ofRPC175 and RPG102.

FIG. 7 is a drawing illustrating a procedure for constructing vector foranalyzing the promoter expression (SEQ ID NO:7-9).

FIG. 8 is a graph showing the results of the analysis on the expressionof RPC175 promoter used in combination with GUS.

FIG. 9 consists of photographs showing an example of the results of theanalysis on the tissue-specific expression of RPC175 promoter in flowerorgans.

FIG. 10 is a graph showing the results of the measurement of theactivity of RPC175 promoter in various rice organs.

FIG. 11 is an illustration showing the procedure for constructing thevector for expressing the protein encoded by RPC175 gene.

FIG. 12 consists of a photograph of SDS-PAGE pattern showing the resultsof the experiment on the expression of the protein encoded by RPC175gene.

FIG. 13 consists of a photograph of Western analysis pattern showing theresults of an experiment on the solubilization of the protein encoded byRPC175.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have conducted an extensive search and, as aresult, discovered the DNA fragment comprising the sequence having thenucleotides of positions 1-1234 in the nucleotide sequence representedby SEQ ID NO:3, a part of this sequence or a sequence derived from thesesequences by deletion, substitution, insertion or addition of one ormore nucleotides and having a promoter activity, thus solving theabove-mention problem.

Thus, in accordance with the present invention, the above mentionedproblem can be solved also by identifying a monocotyledon flowerorgan-specific promoter sequence in a rice genome library with the useof the nucleotide sequence represented by SEQ ID NO:1 as a probe.

Furthermore, the present inventors have solved the above-mentionedproblem by the DNA sequence encoding a chitinase comprising thenucleotides of positions 114-1097 in the nucleotide sequence representedby SEQ ID No:1, a part of said sequence, or a DNA sequence having asequence derived from these nucleotide sequences by deletion,substitution, insertion or addition of one or more nucleotides andencoding a protein having biological activity equivalent to that of theprotein encoded by a DNA consisting of the above-mentioned nucleotidesequence, or a sequence of chitinase consisting of the amino acidsequence represented by SEQ ID NO:2, a part of this sequence or an aminoacid sequence of chitinase having a sequence derived from these aminoacid sequences by deletion, substitution, insertion or addition of oneor more amino acids.

Now, the present invention will be described in greater detail.

As described above, the first invention found by the present inventorrelates to a DNA comprising the sequence having the nucleotides ofposition 1-1234 in the nucleotide sequence represented by SEQ ID NO:3, apart of said sequence or a sequence derived from these sequences bydeletion, substitution, insertion or addition of one or more nucleotidesand having a promoter activity.

The promoter sequence of the present invention, namely, the sequencecomprising the nucleotides of positions 1-1234 in the nucleotidesequence represented by SEQ ID NO:3 has no substantial homology to anyknown promoter sequence. Thus, this sequence is considered to be novel.This sequence can be isolated from a natural monocotyledonous plant inaccordance with the method described in, for example, Example 2 as willbe given hereinafter.

The DNA fragment of the present invention has a promoter activityspecific to flower organs. The term “flower organ-specific promoteractivity” as used herein means that the expression of the promoteractivity the DNA fragment of the present invention in flower organs(anther, filament, pistil and lodicule), in particular in floweringperiod, is more prominent than in other organs (at least leaf, root,callus, germinating seed, immature seed and palea and lemma). It hasbeen confirmed that the DNA fragment of the present invention is flowerorgan-specific in monocotyledons and there is a possibility that it maybe flower organ-specific in other plants too.

The nucleotide sequence represented by SEQ ID NO:3 has the followingcharacteristics among others.

1. It has 3 transcription initiation points at intervals of severalnucleotides and these points are all A (adenine) following TC.Specifically, the transcription initiation points are the adenines (A)at positions 1122, 1125 and 1129.

2. There is a TATA box-like sequence (5′-TATATAA-3′) (Corden et al.Science 209, 1406-1414, 1980) 30 bp upstream of the most upstreamtranscription initiation point.

3. There are 2 ATG sequences in the same reading frame, each beinglocated 77 bp and 113 bp downstream of the most upstream transcriptioninitiation point.

4. A termination codon (TGA) is located 21 bp upstream of the mostupstream ATG (the first ATG). Moreover, there are two poly A signal-likesequences(5′-AATAAA-3′) (Heidecker and Messing, Annu. Rev. PlantPhysiol. 37, 439-466, 1986) in the terminator region. The term“terminator region” herein referrs to the region which is downstream ofthe termination codon.

A SnaBI cleavage site is found between positions 1135 and 1140 while aPstI cleavage site exists between positions 1223 and 1228. The regionfollowing position 1235 is the structural gene region.

It has been found in accordance with the present invention that thepromoter is located in the region upstream of the structural gene, i.e.,in the region comprising the nucleotides of positions 1-1234 in thenucleotide sequence represented by SEQ ID NO:3. However, sequencescomprising a part of this region are also included in the presentinvention, so long as they have a similar promoter activity.

For example, the regions of positions 1-1228 and 1-1140 have the flowerorgan-specific promoter activity, as will be described in the examplesgiven hereinafter. Thus, these sequences are included in the presentinvention. Also, it is expected that the region of positions 1-1121 hasa similar promoter activity, since a transcription initiation point islocated at position 1122 as described above.

Further, it should be noted that the promoter sequence contains an EcoRIsite at nucleotide positions 1-6 by chance, which enabled us todetermine the promoter sequence starting from this site. Therefore, itis well anticipated that a sequence starting from a nucleotide some whatdownstream will have the promoter activity. This is so because a numberof reports indicate that the tissue- or time-specificity or inducibilityof most plant promoters is substantially contained in the region of 0.3to 0.4 kb which precedes the transcription initiation point. In thepromoter of type II glutelin gene of rice, for example, the tissue- andtime-specific expression is fully achieved exclusively by the 441 bpfragment before the transcription initiation point (Takaiwa et al. PlantMol. Biol. 16:49-58, 1991). In the promoter ofself-incompatibility-related gene SLG13 of Brassia oleracea, the 411 bpregion before the transcription initiation point directs the expressionin pistil and pollen (Dzelzkalns et al. The Plant Cell 5:855-863, 1993).In the promoter of anionic peroxidase gene of tomato, theorgan-specificity as well as the pathogen and wound-inducibility aredetermined by the 358 bp region upstream of the transcription initiationpoint (Mohan et al. Plant Mol. Biol. 22:475-490, 1993). Thus, it isobserved for a number of promoters that a part of the reported sequencemaintains the full function if only said part is the region comprisingnucleotides of several hundred bp preceding the transcription initiationpoint.

Accordingly, any DNA sequence obtained from the region within severalhundred bp, preferably about 500 bp upstream of the transcriptioninitiation point and having the flower organ-specificity characterizedin the present invention is included in the present invention. Forexample, if a region within several hundred bp upstream of thetranscription initiation point is easily isolated from rice genome byPCR with the use of primers designed based on the nucleotide sequence ofthe present invention and the region exhibits the flowerorgan-specificity inherent to the promoter of the present invention,then the shorter promoter sequence is included in the present invention.

The present invention further includes in the scope thereof DNAfragments having a sequence derived from these sequences by deletion,substitution, insertion or addition of one or more nucleotides andshowing the promoter activity.

It is well known that when a nucleotide sequence of a DNA having aphysiological activity is slightly modified by deletion, substitution,insertion or addition of one or more nucleotides, the physiologicalactivity of the DNA will be maintained in general. Therefore, thepresent invention includes within the scope thereof DNA sequencesderived from the above mentioned promoter sequence by such slightmodification and having the promoter activity. That is to say, thesequence consisting of the nucleotides of positions 1-1234 in thenucleotide sequence represented by SEQ ID NO:3 parts of this sequencehaving the promoter activity (for example, those consisting of severalhundred bp upstream of the transcription initiation point), and DNAsequences derived therefrom by deletion, substitution, insertion oraddition of a small number of nucleotides and having the promoteractivity are all intended to be included in the scope of the presentinvention.

Similarly, the sequence consisting of the nucleotides of positions1-1140 in the nucleotide sequence represented by SEQ ID NO:3, thesequence consisting of the nucleotide sequence of positions 1- to 1121thereof, and DNA sequences derived therefrom by deletion, substitution,insertion or addition of a small number of nucleotides and having thepromoter activity are all included in the scope of the presentinvention.

The addition, insertion, deletion or substitution of nucleotides can becarried out by, for example, site-directed mutagenesis (see, forexample, Nucl. Acids Res. 10:6487-6500, 1982) which is a well-knowntechnique. The expression “one or more nucleotides” as used herein meansnucleotides in such a number as to allow addition, insertion, deletionor substitution by the site-directed mutagenesis method.

Site-directed mutagenesis can be performed in the following manner withthe use of, for example, a synthetic oligonucleotide primer which iscomplementary to the single-stranded phage DNA to be mutated except aspecific discordance, i.e., the desired mutation. Namely, acomplementary strand is synthesized by a phage with the use of theabove-mentioned oligonucleotide as a primer. Next, a host bacteriumcarrying the phage is transformed by the double-stranded DNA thusobtained. The culture of the transformed bacterium is then plated ontoagar and plaques containing the phage from a single cell are formed.Thus theoretically 50% of the newly formed colonies will contain thephage carrying the mutation in the single strand while the remaining 50%of the colonies have the original sequence. The plaques thus obtainedare hybridized with a synthetic probe having been treated with kinase atsuch a temperature as to allow the hybridization of the plaquescoinciding with the DNA having the desired mutation as described abovebut not with those having the original strands. Then the plaqueshybridized with the probe are picked up and cultured to subsequentlyrecover the DNA.

In addition to the above site-directed mutagenesis method, nucleotide(s)can be substituted, deleted, added or inserted into the promotersequence while maintaining its activity by treating the gene with amutagen or by selectively cleaving the gene and then deleting, adding orsubstituting the desired nucleotide(s) followed by ligation.

Also, the substitution, deletion, addition or insertion of specificnucleotide(s) may be conducted by the site- directed mutagenesis withthe use of the PCR method (Mikaelian et al. Nucl. Acids Res. 20:376,1992) or the random nucleotide substitution technique (Zhou et al. Nucl.Acids Res. 19:6052, 1991) by taking advantage of the low fidelity of TaqDNA polymerase.

Now, the second invention found by the present inventors will beillustrated.

The second invention of the present invention relates to a monocotyledonflower organ-specific promoter sequence which is contained in a sequenceidentified from a rice genome library with the use of the nucleotidesequence represented by SEQ ID NO:1 as a probe.

This nucleotide sequence represented by SEQ ID NO:1 can be obtained byconstructing a CDNA library from rice (Oryza sativa) palea and lemma orpistil, isolating a CDNA which is expressed specifically in pistil,anther and lodicule by differential screening and determining the wholenucleotide sequence thereof. The nucleotide sequence thus determined maybe used as a probe as a whole. Alternatively, use may be made of a partthereof as a probe. In this case, any hybridization and washingconditions are employable so long as they enable the formation of amolecular hybrid if the nucleotide sequence of the DNA to be identifiedhas a homology of 80% or more to the nucleotide sequence represented bySEQ ID NO:1.

The genome library of rice is constructed by using rice (Oryzae sativat)green leaf, though the present invention is not limited thereto. Thepromoter sequence is identified from the library thus obtained by usingthe above-mentioned probe. In order to determine that the promoter isspecific to flower organs, a chimera gene is constructed by ligatingβ-glucuronidase (GUS) gene to the promoter sequence. The resultantchimera gene is introduced into rice plant and then the expression sitesare confirmed.

The determination of the above-mentioned nucleotide sequence representedby SEQ ID NO:1, the construction of the rice genomic library and theassessment of the specific promoter activity are described in detail inExamples as will be given hereinafter, though the present invention isnot limited thereto.

As described above, the nucleotide sequence represented by SEQ ID NO:3,in particular, the sequence comprising the nucleotides of positions1-1234 is a novel DNA fragment isolated by the present inventors. Basedon the disclosure of the present invention, those skilled in the art caneasily isolate DNA fragments having a flower organ-specificity similarto the one of the present invention from various monocotyledon genomelibraries with the use of at least a part of the nucleotide sequencecomprising the nucleotides at positions 1-1234 of the nucleotidesequence represented by SEQ ID NO:3. The conditions for hybridizationwith the probe can be appropriately determined too. Therefore, thepresent invention includes within the scope thereof DNA fragments whichare hybridizable with at least a part of the nucleotide sequenceconsisting of the nucleotides of positions 1-1234 of SEQ ID NO:3 andhave a flower organ-specific promoter activity similar to that of thepresent invention.

The promoter of the present invention is a novel flower organ-specificpromoter which makes it possible to genetically manipulate and improvenot only anther but also pistil and lodicule which was previouslyimpossible particularly in monocotyledons. Thus the promoter is usefulfor, e.g., the following purposes.

(1) Creation of female sterile plants by use of a structural genecapable of inducing sterility wherein said gene is ligated to thepromoter sequence of the present invention or a part thereof.

(2) Flower organ-specific enlargement or elongation by use of astructural gene capable of promoting the elongation or division of plantcells wherein said gene is ligated to the promoter sequence of thepresent invention or a part thereof.

(3) Genetic regulation of flowering by means of the expression of thepromoter of the present invention in lodicule.

(4) Providing the whole flower organs or a part thereof with an improvedtolerance to herbicides or diseases by use of a gene imparting toleranceto herbicides or resistance to diseases wherein said gene is ligated tothe promoter.

The promoter of the present invention is expressed in the stigma, style,anther wall, filament and lodicule of rice in the flowering period. Whenthis promoter is used, for example, in the improvement of male sterilerice, its expression in anther wall and filament can be ignored.Further, it is sometimes expected that the sensitivity to a gene productvaries from organ to organ. In such a case, the promoter of the presentinvention will be useful, for example, to specifically improve stigmaand style or lodicule.

Finally, the third invention established by the present inventors willbe illustrated.

The third invention relates to a DNA sequence which comprises thenucleotides of positions 114-1097 in the nucleotide sequence representedby SEQ ID NO:1 or a part of said sequence, or a DNA sequence derivedtherefrom by deletion, substitution, insertion or addition of one ormore nucleotides and encoding a protein having a biological activityequivalent to that of the protein encoded by the DNA consisting of theabove-mentioned nucleotide sequence. The third invention further relatesto a sequence consisting of the amino acid sequence represented by SEQID NO:2, a part of this sequence or an amino acid sequence derived fromthese amino acid sequences by deletion, substitution, insertion oraddition of one or more amino acids and having a biological activityequivalent to that of the protein consisting of the above-mentionedamino acid sequence.

The DNA sequence of the present invention, i.e., the sequence consistingof the nucleotides of positions 114-1097, and the amino acid sequencerepresent a novel chitinase having homologies of 67 to 69% and 54 to61%, respectively to the known rice class I chitinase.

The amino acid sequence represented by SEQ ID NO:2 has the followingcharacteristics.

By analogy based on the probable homologies to various class Ichitinases, its structure is supposed to have the following elementsfrom the N-terminal side thereof: a leader sequence having consecutivehydrophobic amino acid residues (amino acids of positions 1-20 in SEQ IDNO:2) at the N-terminus; a chitin-binding region rich in cysteineresidues (amino acids of positions 21-61 in SEQ ID NO:2) in theN-terminus region of the mature protein; and a spacer region (aminoacids of positions 62-83 in SEQ ID NO:2) followed by the catalyticregion (amino acids of positi84-328 in SEQ ID NO:2). In this catalyticregion, the first tyrosine residue (Verburg et al. J. Biol. Chem., 267,3886-3893, 1992; Y at position 199 in SEQ ID NO:2) in NYNYG (amino acidsat positions 198-202 in SEQ ID NO:2), which is considered as the activesite of chitinase, is conserved. At the C-terminus, characteristicconsecutive amino acid residues (at positions 318-328 in SEQ ID NO:2)showing no homology to other rice chitinases are observed. The molecularweight and isoelectric point of the mature protein region (frompositions 21 to 328 in SEQ ID NO:2) are calculated respectively as about32 kD and 7.24.

When mature protein region (amino acids of positions 21-328 in SEQ IDNO:2) is expressed in E. coli and the chitinase activity is measured asone of the biological activities thereof, the chitinase activity can bedetected in practice.

As discussed above, a plant class I chitinase has a cysteine-richchitin-binding region which is followed by a spacer region at theN-terminus of the mature protein. However, Iseli et al. (Plant Physiol.10221-226, 1993) reported that the chitinase and antimicrobialactivities are maintained in the absence of these regions. Accordingly,it is highly probable that the chitinase of the present invention alsohas chitinase activity exclusively in the catalytic region, namely,without the chitin-binding and the spacer regions. Therefore, it isexpected that the catalytic region consisting of the amino acids ofpositions 84-328 in SEQ ID NO:3 should have a biological activityequivalent to those of the entire 175 protein (amino acids of positions1-328 in SEQ ID NO:2) or the mature protein (amino acids of positions21-328 in SEQ ID NO:2). Thus, this region is also included in thepresent invention.

As described above, the novel chitinase of the present invention haschitinase activity and thus is useful for the following purposes:

(1) Production of disease or insect damage-resistant plants by way ofthe transformation of plant cells by the DNA sequence comprising thenucleotides of positions 114-1097 in the DNA sequence represented by SEQID NO:1 of the present invention or a part thereof, wherein said DNAsequence or a part thereof has been ligated to a constitutive,tissue-specific, time-specific or inducible promoter.

(2) Application of chitooligosaccharides produced by the chitinase asmaterials for manufacturing foods, cosmetics and drugs.

EXAMPLES Example 1 Isolation of Flower Organ-specific cDNA

Paddy rice varieties “Akihikari”, “Tsukinohikari” and “IR24” were grownin a greenhouse and subjected to the following experiments.

(1) Extraction of RNA

The leaf, pistil, anther, lodicule, palea and lemma, immature seed,germinating seed, root and callus of “IR24” and palea and lemma (4.5 to6.0 mm in length) of “Akihikari” were collected, immediately frozen inliquid nitrogen and then stored at −80° C. A portion of the pistil wasdivided into the stigma and ovary tissues.

The total RNA was extracted from these tissues by the SDS-phenol methodof Watanabe and Price (Proc. Natl. Acad. Sci. USA, 79, 6304-6308, 1982)except that β-mercaptoethanol was added as an antioxidant to theextraction buffer to give a final concentration of 10% (V/V). Thetissues to be used in the reverse transcription PCR experiment weretreated with DNase I (FPLC pure, manufactured by Pharmacia) in thepresence of RNase inhibitor (RNAguard, manufactured by Pharmacia),rather than being subject to lithium chloride precipitation, so as tominimize the contamination with any trace amount of DNA. The leaf androot [expressed in “root (soil)” in FIG. 2 and Table 1 as will be givenhereinafter] were collected from a plant grown for 1 month in thegreenhouse after sowing. The pistil, anther, lodicule and palea andlemma were collected from a plant immediately to several days beforeflowering. The immature seed was collected from a plant 1 to 2 weeksafter flowering. The germinating seed and root were obtained from aplant aseptically grown on an N6 medium (Chu et al. Scientia Sinica, 18,659-668, 1975) respectively for 1 and 3 weeks after sowing.

The callus was induced from a seed in an N6 solid medium containing 2mg/l of 2,4-D and then cultured before use in a liquid medium of thesame composition under shaking for 1 week. The total RNA of the pistiland leaf was purified to provide polyA+RNA by using Oligotex-dT30 super(manufactured by Takara Shuzo Co., Ltd.) in accordance with themanufacturer's instructions

(2) Construction of Palea and Lemma and Pistil cDNA Libraries

About 2.2 μg and 1 μg of respective polyA+RNA isolated and purified frompalea and lemma and pistil was employed as a template to synthesize thecDNA by using ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE). Thedetermination of RI uptake ratio indicated that about 462 ng and about55 ng of the first strand cDNA of the palea and lemma and pistil werereversely transcribed by the oligo-dT priming, and about 1,022 ng andabout 72 ng of the second strand cDNA were synthesized directly from thefirst strands. In accordance with the manufacturer's instructions, thecDNA was connected to an EcoRI adaptor and digested with XhoI, to beligated into vector Uni-ZAPXR. Next, the phage DNA was packaged intophage particles by using Giga-pack Gold packaging extract (manufacturedby STRATAGENE). The phage was transfected into E. coli PLK-F′ hostcells, which were then inoculated on a plate and each library size wasexamined. As a result, the palea and lemma CDNA library size wascalculated as 1×106 pfu (plaque forming unit) while that of the pistcDNA library was calculated as 3×10⁶ pfu.

(3) Differential Screening

Differential screening was carried out basically in accordance with themethod of Gasser et al. (The Plant Cell 1, 15-24, 1989). About 2,000 pfuof the phage from the palea and lemma cDNA library was infected into E.coli PLK-F′ cells and the cells were plated on square petri dishes(14×10 cm). For each plate, a replica filter was prepared with the useof a nylon membrane filter Hybond-N+(manufactured by Amersham) and thefilter was treated in accordance with the manufacturer's instructions.

As the probes for hybridization, use was made of single-stranded cDNAsynthesized from about 100 ng of the polyA+RNA (or about 2 μg of thetotal RNA) of pistil and leaf. To 2 μl of an RNA solution, 0.5 mM ofd(ATG)TP, 10 mM of DTT and 133 M-MuLV buffer (manufactured by BRL) wereadded. Next 30 ng/μl of Random DNA Hexamer (manufactured by Pharmacia)[or 80 ng/μl of Oligo dT Primer (manufactured by Amersham)] was addedthereto (the concentration indicates the final concentration in eachcase). After dissociating the secondary structure of the RNA by heatingat 65° C. for 5 minutes, the primer was annealed at room temperature.After further adding 1.5 unit/μl of RNase inhibitor (RNAguard,manufactured by Pharmacia), 10 unit/μl of reverse transcriptase M-MuLV(manufactured by BRL) and 4 μCi/μl of [α-³²P]dCTP (each expressed in thefinal concentration), the liquid reaction mixture of 20 μl in total wasmaintained at 37° C. for one hour. Subsequently, RI-unlabeled dCTP wasfurther added to give a final concentration of 0.5 mM and the reactionwas continued for 30 minutes.

The labeled DNA probes were purified by using Quick Spin Column G-50Sephadex (manufactured by BOEHRINGER MANNHEIM). The probes weredenatured by adding an equivalent amount of 2 N NaOH (finalconcentration: 1 N). The filter was first treated in a hybridizationbuffer (0.25 M Na₂HPO₄ pH 7.2, 7% SDS, 1 mM EDTA, 133 Denhardt'ssolution) at 68° C. for 10 minutes. Then the single-stranded probes(final concentration: 0.2-0.3×10⁷ cpm/ml) and carrier.DNAs (0.1 mg/ml,salmon sperm DNA, 0.1 μg/ml λDNA, 0.1 μg/ml rice DNA) were added theretoand hybridization was performed at 68° C. overnight (16 to 24 hours).

The filter was washed in the buffer (20 mM Na₂HPO₄ pH 7.2, 1% SDS, 1 mMEDTA) at room temperature twice and at 68° C. twice each for 15 minute.Next, this filter was exposed to Kodak X-Omat Film at −70° C. for 4 to 5days. When about 20,000 plaques were examined, 114 plaques showingintense hybridization signals with the pistil probe but only weak orbackground signals with the leaf probe were selected by the primaryscreening. Subsequently, these plaques were further purified and 41plaques were selected in the tertiary screening stage.

Among these plaques, one showing a particularly weak signal with theleaf probe (indistinguishable from the background) was stored in 200 μlof SM buffer (0.1 M NaCl, 7 mM MgSO₄, 50 mM Tris-CL, pH 7.5, 0.01%gelatin) containing one drop of chloroform at 4° C. Then the thus storedliquid was diluted and the phage was plated so as to give a considerablylow plaque density (10 to 100 pfu/plate). A plaque separated from otherswas isolated and stored in the same buffer. From this liquid, a lysate(plating lysate) containing the phage at a high concentration wasprepared and in vivo excision was performed in accordance with theinstructions attached to ZAPcDNA Synthesis Kit. Thus a plasmid[pBluescriptSK(−)] was cut from the phage genome. Then it was digestedwith restriction enzymes EcoRI and XhoI (manufactured by Takara ShuzoCo., Ltd.) and thus a cDNA insert (about 0.8 kb) was isolated andpurified.

(4) Analysis on Organ-specific Expression of cDNA Clones

i) Northern Hybridization Analysis

The cDNA clone selected in the above (3) was subjected to Northernhybridization to examine the expression patterns and expression levelsin various organs. Filters were prepared in the following manner. First,the secondary structure of the total RNA (20 μg) from each of the organsdescribed in the above (1) was dissociated in accordance with the methodof Sambrook et al. (Molecular Cloning, 1982) with the use of deionizedGlyoxal and DMSO and then fractionated in a 1% agarose gel. Next, theRNA was blotted onto a nylon membrane Gene Screen Plus (DU PONT) by theconvention capillary transfer method. After drying in a vacuum oven at80° C. for 1 hour, the filter was boiled in 20 mM Tris-Cl (pH 8.0) for 5minutes to thereby remove Glyoxal therefrom. As a probe, the 0.8 kbEcoRI fragment of the above-mentioned cDNA was RI-labeled by usingMultiprime Labeling System (manufactured by Amersham).

Pre-hybridization and hybridization were carried out in accordance withthe manufacturer's instructions attached to the filter. The filters werewashed with 2×SSC, 1% SDS and 0.2×SSC, 1% SDS at room temperature eachfor 5 minutes, then with 0.16×SSC, 1% SDS at 65° C. for 15 minutes twiceand then with 2×SSC at room temperature for 1 minute. Subsequently thefilters were exposed to Kodak X-Omat Film at −70° C. overnight. As aresult, signals were observed exclusively in the lanes of pistil, antherand lodicule while the other lanes showed no signal, as shown in FIG. 1.Thus, it was clarified that the differential clone isolated above wasexpressed strongly in pistil, anther and lodicule but in very orextremely low level in other organs. The size of the transcripts wasestimated to be 1.5 kb.

Then the expression doses were determined by measuring the signaldensities with a densitometer. As a result, the relative expressionlevels in anther and lodicule were respectively about 2 and about 4,taking that in pistil as 1.

ii) Reverse Transcription PCR Analysis

To analyze the organ-specific expression of the cDNA clone at a higherprecision, reverse transcription PCR was carried out by using RNA ofvarious rice organs as templates. First, the partial nucleotide sequenceof the cDNA was determined. By using GENESIS 2000 Fluorescence Sequencer(manufactured by Du Pont), the nucleotide sequence of the cDNA insertedinto the plasmid pBluescript SK(−) was determined. In accordance withthe manufacturer's instructions attached to the Sequencer, T7 DNApolymerase reaction was performed by using RV and M4 primers(manufactured by Takara Shuzo Co., Ltd.) of M13 followed byelectrophoresis on 6% acrylamdie gel. Then, the nucleotide sequence wasdetermined from both the 5′- (EcoRI) an3′-(XhoI) sides.

On the basis of about 300 nucleotides in the 5′-side of this DNA,appropriate primers:

5′-GACACCCGCAAGCGTGA-3′ (75RV1 (SEQ ID NO:10): 17-mer) and

5′-CCCTTCACCTCCTTGTA-3′ (75FW1 (SEQ ID NO:11); 17-mer);

were synthesized with DNA Synthesizer (manufactured by ABI) and purifiedby OPC Cartridge (manufactured by ABI) for the reverse transcription PCRexperiment. With these primers, a product of 101 bp was amplified.

Further, the following primers synthesized based on the sequence of riceactin 1 gene (Racl, McElroy et al. Plant Mol. Biol. 14, 163-171, 1990

5′-GTATCCATGAGACTACATACAACT-3′ (24-mer) (SEQ ID NO:12) and

5′-TACTCAGCCTTGGCAATCCACA-3′ (22-mer) (SEQ ID NO:13);

were used as controls. These primers were selected so that an intronwould be included therebetween. Therefore, it was intended that if thetemplate DNA was contaminated with genomic DNA, a product (350 bp) ofthe genomic DNA would be amplified along with the product (267 bp) ofthe cDNA The total RNA of each of the above-mentioned organ was seriallydiluted (1 μg, 30 ng, 1 ng, 30 pg) and treated at 65° C. for 5 minutesto thereby dissociate the secondary structure. After quenching on ice,it was incubated in a reaction mixture comprising 1×Perkin Elmer GeneAmpbuffer 1 mM dNTPmix, 5 ng/μl oligo dT15 primer (manufactured byAmersham), 2.5 mMgCl₂, 3 U/μl RNase inhibitor (RNAguard, manufactured byPharmacia) and 1 U/μl reverse transcriptase M-MuLV (manufactured by BRL)(each concentration referrs to the final concentration) at 37° C. for 30minutes. Next, it was treated at 95° C. for 5 minutes to dissociate theRNA-CDNA hybrid and then cooled on ice. Then a pair of primers (10-20pmoles), 10×buffer and 1 unit of AmpliTaq Polymerase (manufactured byPerkin Elmer) were added and the reaction mixture of 50 μl in totalvolume was subjected to PCR for 40 cycles with each cycle consisting of1 minute at 94° C., 1 minute at 60° C. and 2.5 minutes at 72° C.

As a control experiment, the reverse transcription product (i.e., totalRNA) prepared from each organ was subjected to PCR with the use ofprimers for Racl gene, which is considered to be constitutivelyexpressed in all organs in rice. As a result, the anticipated PCRproduct (267 bp) was detected from all of the organs examined. Thisindicated that no template cDNA preparations employed were contaminatedwith substantial amount of genomic DNA. The detection limits (amount oftemplate RNA) in germinating seed and root were 0.5 μg and 1 ngrespectively, while the limits in other organs were 30 ng. Supposingthat Rac1 gene was expressed in all of the organs tested herein at thesame level, it was estimated that the reverse transcription PCRefficiencies in germinating seed and root were respectively about 1/17and about 30, taking that in pistil as 1, while those in other organswere almost the same as that in pistil.

Subsequently, a reverse transcription PCR experiment was carried out byusing clone-specific primers which had been preliminarily proved toamplify the product of expected molecular weight by using plasmidclones. When the CDNA reversely transcribed from 1 μg of RNA was used asa template in PCR, the expected product (101 bp) was detected frompistil, anther, immature seed and palea and lemma, as shown in FIG. 2A.Namely, no PCR product was amplified from cDNA of other organs. In thecase of pistil, the expression was observed both in stigma and ovary. Ineach organ from which the PCR product was detected, the template RNA wasdiluted and the expression level was estimated. As a result, in pistil(stigma and ovary) and anther, the PCR product was amplified from themost minimum quantity of the template (30 pg). In contrast, the PCRproduct was amplified from 30 ng or above, and 1 μg of the template inpalea and lemma, and immature seed, respectively (FIG. 2B).

When the expression level in pistil (stigma and ovary) was taken as 1,therefore, the level in anther was estimated as about 1 while those inpalea and lemma and immature seed were calculated respectively as 10⁻³and 3×10⁻⁵. That is to say, this gene was expressed only at an extremelylow level in organs other than flower organs. Table 1 summarizes theresults of the reverse transcription PCR and the Northern analysis.

TABLE 1 Relative expression dose of RPC175 in various organs estimatedfrom Northern analysis and reverse transcription PCR (referring the dosein stigma or pistil as to 1) Organ pistil (stigma ovary) anther lodiculeleaf Northern 1 (NT NT) 2 4 0 analysis RT-PCR 1 (1 ≦1) 1 NT 0 rootgerminating Organ root (soil) seed callus Northern 0 NT 0 0 analysisRT-PCR 0 0 0 0 Organ palea and lemma immature seed Northern  0   0analysis RT-PCR 10⁻³   3 × 10⁻⁵ NT: not analyzed.

(5) Genomic Southern Hybridization Analysis

One month after sawing, genomic DNA was isolated and purified by thephenol SDS method (Komari et al. Theor. Appl. Genet. 77, 547-552, 1989)from paddy rice plants of the varieties of “Tsukinohikari” and “IR24”.About 5 μg of DNA was digested with restriction enzymes BamHI, EcoRI,HindIII, PstI, SalI and XhoI (manufactured by Takara Shuzo Co., Ltd.)and the DNA fragments were fractionated in a 0.8% agarose gel. Afterblotting the DNA onto a nylon membrane filter HybondN+ (Amersham),genomic Southern hybridization was carried out with the use of theabove-mentioned cDNA fragment of 0.8 kb which had been RI-labeledsimilar to the above (4)i).

The hybridization and the subsequent washing were effected according tothe manufacturer's instructions attached to the filter. As a result,several faint bands were observed in addition to one or two intensebands (FIG. 3), though the hybridization was effected under conditionsthat would not allow any hybridization to take place unless the genomicDNA had a high homology to the probe. That is to say, when digested withEcoRI, for example, a strong signal (2.6 kb) and three weak signals of1.6 kb were detected. It was considered that such a faint band mighthave a somewhat short homologous region to the probe or might not havehigh homology to the probe. These results indicate that the cloned genemay have a few related sequences in rice genome.

(6) Isolation of Full-length cDNA

The pistil cDNA library [Example 1 (2)] was screened with the use of thecDNA clone of 0.8 kb as a probe. About 1.6×10⁵ pfu of the phagecontaining the pistil CDNA was plated on 8 square petri dishes in thesame manner as that of Example 1 (3) and replica filters were prepared.These filters were subjected to hybridization with the above CDNA (0.8kb) which had been RI-labeled with the use of Multiprime Labeling System(manufactured by Amersham). As a result, 40 positive plaques wereobtained by the primary screening. Among these plaques, 20 weresubjected to in vivo excision and plasmids containing the cloned cDNAwere cut out from phage DNA.

Subsequently, these plasmids were digested with EcoRI and the cDNAclones were excised. When these clones were compared with each other,the longest ones (3 clones) were about 1.25 kb in size. One typicalclone was selected therefrom and the nucleotide sequence of about 300 bpat the 3′-side was determined. When compared with the correspondingregion of the clone of 0.8 kb from “Akihikari” employed as a probe, thenucleotide sequences of these clones almost completely coincided witheach other including the 3′-untranslated region.

To examine in detail whether or not the obtained clone (1.25 kb) and theclone (0.8 kb) originated from the same gene, a pair of primers asfollows were synthesized based on the nucleotide sequence determinedabove:

5′-GACATCATGTCGGCGTCTGCG-3′ (175RV1 (SEQ ID NO:14); 21-mer) and

5′-GCCATGACCATGCATACATATGG-3′ (175FW1 (SEQ ID NO:15); 23-mer).

Then reverse transcription PCR was effected for all of the rice organsin the same manner as the one employed in Example 1 (4) ii) but for 30cycles. The results thus obtained were the same as those obtained in thecase of the clone of 0.8 kb.

Accordingly, it was clarified that the gene of the present invention wasexpressed almost exclusively in pistil, anther and lodicule. Theexpression level thereof in palea and lemma and immature seed were about1/1,000 times as much as the expression level in pistil, anther andlodicule, while no detectable level of expression was observed in otherorgans. These facts clarified that the selected cDNA clone of 1.25 kbhad the same origin as that of the cDNA of 0.8 kb isolated by thedifferential screening. Thus, this cDNA clone was employed as the probein the subsequent isolation of genomic clones. This cDNA clone of 1.25kb was named “RPC175”.

(7) Determination of the Nucleotide Sequence of RPC175

The entire nucleotide sequence of the cDNA clone RPC175 (about 1.25 kb),which is expressed specifically in flower organs, was determined in thefollowing manner with the use of Fluorescence Sequencer (Model 373A,manufactured by Applied Biosystems). By using the M13 primers RV(5′-side of the cDNA, decoding the sense strand) and M4 (3′-side of thecDNA, decoding the antisense strand) on the plasmid vector pBluescriptSK(−) carrying RPC175 integrated thereinto, the nucleotide sequences ofabout 250 to 300 bp in the 5′- and 3′-sides were first determined.

Based on the nucleotide sequence information thus obtained, internalprimers were constructed and the nucleotide sequences of the sensestrand and antisense strand were decoded from both sides. Next, furtherprimers were synthesized based on the partial nucleotide sequences thusdecoded and the nucleotide sequences of the sense strand and antisensestrand were consecutively decoded. Thus, 9 internal primers (4 for thesense strand and 5 for the antisense strand) were used in totalincluding the primers used in the reverse transcription PCR. On theother hand, it was known by restriction analysis that RPC175 had twoApaI sites. Thus, RPC175 was split into 3 fragments at these sites andsubcloned into the same site of the plasmid vector pBluescript SK(−).Then the nucleotide sequence was determined by the M13 primers.

Thus the entire nucleotide sequence of RPC175 was determined by usingthe internal primers and the subcloning. Next, the nucleotide sequencethus determined was analyzed by using GENETYX-MAC8.0, a software foranalyzing nucleotide sequence/amino acid sequence. As a result, it wasfound that the complete nucleotide sequence of RPC175 consisted of 1,258bp and had 2 initiation codons (ATG), separated by a sequence of 36 bp,in the same reading frame in the 5′-side and the upstream ATG waslocated immediately after a sequence capable of forming a stem loop.

The reading frame as determined by the ORF analysis enabled estimationof amino acids. Thus, it was determined that 340 amino acids wereencoded when the translation was initiated from the upstream ATG, while328 amino acids were encoded when the translation was initiated from thedownstream ATG. Two polyA signals (5′-AATAAA-3′) were located downstreamof the termination codon. The entire nucleotide sequence of RPC175 isrepresented by SEQ ID NO:1. It is to be understood that the first 60 bpregion originate in the genome clone as will be described hereinafterand that SEQ ID NO:1 includes all the nucleotides from the transcriptioninitiation point to polyA.

The amino acid sequence encoded by RPC175 is represented by SEQ ID NO:2.As will be described hereinafter, it was highly probable that of the twoATGs the downstream ATG was the transcription initiation point. Thus,the amino acid sequence consisting of 328 amino acids in total from thedownstream ATG to the termination codon is shown in SEQ ID NO: 2. In theamino acid sequence represented by SEQ ID NO:2, the sequence consistingof the amino acids of positions 1-20 is estimated to be the leadersequence while the sequence after the leader is the amino acid sequenceof the mature protein, as will be illustrated hereinafter.

A homology analysis was conducted by the software GENETYX-MAC/CD 32 andBLAST, an internet program for nucleotide sequence detection. As aresult, RPC175 was homologous to class I chitinases of rice, wheat,barley, corn, potato and tomato. That is, it showed homology over theentire regions, excepting for the variable region (amino acids ofpositions 62-83 in SEQ ID NO:2), including the leader sequence withconsecutive hydrophobic amino acids (positions 1-20), the chitin-bindingregion rich in cysteine (positions 21-61) and the catalytic region(positions 84-328). The catalytic region contained the first tyrosineresidue in NYNYG (Y at position 199 in the amino acid positions 198-202positions in SEQ ID NO:2) which is conserved in a number of basicchitinases and considered as the active site. At the C-terminus,characteristic consecutive amino acids (amino acids positions 318-328 inSEQ ID NO:2) were observed unlike other rice chitinases. RPC175 showedhomologies to various rice class I chitinases of about 67 to 69% basedon the nucleic acids and about 54 to 61% based on the amino acids.

The molecular weight and isoelectric point of the mature protein (aminoacids of positions 21-328 in SEQ ID NO:3) were calculated respectivelyto be about 32 kD and 7.24.

Example 2 Isolation of Promoter

(1) Construction of Genomic Library

Genomic DNA was isolated by the SDS-phenol method, and purified by thelithium chloride precipitation method for elimination of RNA from riceleaves about 2 months after sowing. As a preliminary test, the DNA wasfirst partly digested with a restriction enzyme MboI (manufactured byTakara Shuzo Co., Ltd.) to determine the digestion conditions whichwould allow the formation of fragments of 16 to 23 kb in apparent size.Next, the genomic DNA was digested under the so determined reactionconditions and subjected to sucrose density gradient centrifugation.Sucrose was dissolved in a buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 200mM NaCl) to give a gradient of 5 concentrations (10, 17.5, 25, 32.5 and40%). These sucrose solutions were layered in this order in acentrifugation tube (40PA, manufactured by Hitachi) and finally thepartly digested DNA solution was layered on top of the gradient. Aftercentrifuging at 20,000 rpm for 17 hours at 20° C. by using a rotor SRP28SA (manufactured by Hitachi), the mixture was divided into 80 portions(0.5 ml each) with a peristaltic pump (AC-2110, manufactured by Atto) toprovide a fraction containing DNA fragments of 16 to 23 kb in thelargest amount.

This DNA fraction was then ligated with a vector LAMBDA DASHII/BamH(manufactured by STRATAGENE) by the action of T4 DNA ligase(manufactured by BOEHRINGER MENNHEIM) and then packaged into phageparticles by using Gigapack II Gold packaging extract (manufactured bySTRATAGENE). Thus, a rice genomic library was constructed, the size ofwhich was calculated as about 5×10⁶ pfu.

(2) Screening of Clones

About 10,000 pfu of the phage was mixed with E. coli SRBP2 for infectionand inoculated into a square petri dish (14×10 cm). After an incubationat 39° C. overnight, a nylon membrane filter Hybond N+ (manufactured byAmersham) was brought into contact with the plaque surface and thenprocessed in accordance with the manufacturer's instructions attached tothe filter. The probe was a 1.2 kb EcoRI fragment of the rice flowerorgan-specific cDNA (RPC175) which was used after being RI-labeled withthe use of Multiprime Labeling System (manufactured by Amersham). Thus,plaque hybridization was carried out. The hybridization and washing wereeffected under the same conditions as those specified in the aboveExample 1 (3) provided that 1×Denhardt's solution and carrier DNAs werenot employed. From about 160,000 plaques, 35 positive clones wereselected in the primary screening. Subsequently, the secondary screeningwas performed to give 12 positive clones.

Next, phage DNAs were prepared from these plaques. They served astemplates, in the PCR which was performed with the use of theRPC175-specific primers 175FW1 and 175RV1 as in the foregoing reversetranscription PCR. From the PCR experiment using gene-specific primers,the target clones were screened. As a result, the expected product ofabout 200 bp was found to have been amplified in 8 clones out of 12.Five clones among them were further subjected to PCR by using anotherset of primers (75FW1 and 75RV1). As a result, a product longer by about90 bp than the one amplified by using cDNA as a template was amplifiedin every case.

Subsequently, the nucleotide sequence was determined for the PCRproducts of these 5 clones at 2 sites (about 400 bp in total). Whencompared with the nucleotide sequence of the control cDNA, these 5genomic clones all showed a homology of 99% or above except the intronsequence. Based on these facts, it was concluded that these clones mostprobably represented the genomic clone which was the target of thisscreening.

The product obtained by using 75FW1 and 175RV1 had an intron of 85 bphaving a 5′-GT-AG-3′ sequence in the both ends thereof. Therefore, whenthe DNA of the genomic clone was employed as a template, a PCR productlonger than that amplified by using cDNA as a template was amplified.This intron had a PstI site at the 3′-terminus.

(3) Subcloning of Gene Region

The total genomic DNA of rice was digested with a restriction enzymeEcoRI and genomic Southern analysis was carried out by using the RPC175gene as a probe. Thus a band with a weak signal appeared at about 1.6 kbin addition to the one with a strong signal at about 2.6 kb (FIG. 3). Onthe other hand, phage DNA was extracted from the above-mentioned 5clones and digested with EcoRI followed by Southern hybridization withthe use of RPC175 as a probe. As a result, it was found that the DNAfragments which formed hybridization with RPC175 were limited to thoseof 2.6 kb and 1.6 kb, which agreed with the results of the Southernanalysis on the genomic DNA. It was known from the nucleotide sequencedata, that RPC175 had the unique EcoRI site about 70 bp upstream of the3′-terminus. Therefore, the 1.6 kb fragment with a weak signal wasconsidered to have been detected due to the homology between the shortregion (about 70 bp) from the EcoRI to the poly A sites in the 3′-regionof RPC175 cDNA employed as a probe and the genomic DNA fragment.

From the signal intensity in the genomic Southern analysis, it wasanticipated that the 2.6 kb EcoRI fragment would include the completestructural gene region and at least about 1 kb upstream thereof, unlessit contained a large intron. Thus, this fragment was subcloned into theplasmid vector Bluescript SK(−) and named “RPG102”. For furtheranalysis, RPC102 was digested with a restriction enzyme PstI andelectrophoresed on an agarose gel, whereby, RPC102 was divided into 4fragments of about 1.2, 0.8, 0.4 and 0.2 kb.

Next, these DNA fragments were transferred onto a filter and subjectedto Southern analysis with the use of RPC175 as a probe. As a result,signals were detected in 3 (about 0.8, 0.45 and 0.1 kb) out of the 4fragments. It was known from the nucleotide sequence data that RPC175had a PstI site about 45 bp downstream of the 5′-terminus and anotherPstI site about 120 bp downstream thereof. Thus, the band of 0.1 kb insize detected by the Southern analysis was assignable to this region. Itwas also known that another PstI site was located about 50 bp upstreamof the EcoRI site at the 3′-terminus, and in addition, an analysis withthe use of restriction enzymes indicated that the distance between thesecond PstI site in the 5′-side and the PstI site in the 3′-side wasabout 0.95 kb. Accordingly, it was assumed that RPG102 contained anintron of about 200 bp in addition to the above-mentioned intron of 85bp, and that the fragment of 1.25 kb was cut into the fragments of 0.8kb and 0.45 kb at the PstI site in the intron of 85 bp.

Based on these facts, it was considered that the 3 bands detected by theSouthern analysis corresponded to the structural gene region and thatthe promoter region was contained in the PstI fragment of 1.2 kbp whichdid not form a molecular hybrid with RPC175 cDNA.

(4) Identification of Promoter Region

By analyzing the nucleotide sequence in the 5′-side of RPC175 cDNA, itwas clarified that two ATGs were contained in the same reading frame inthis region of RPC175.

A primer containing the downstream ATG:

5′-CTTCATGGCCACCTGCAGGTTTGC-3′ (C5FW (SEQ ID NO:16); 24-mer) wassynthesized and the nucleotide sequence of about 300 bp in the 3′-sideof the promoter region of RPG102 was determined.

To ensure the determination of the transcription initiation point by theprimer extension method, another primer of about 40 bp upstream of C5F

5′-TGCGATCATGGCAAGATGC-3′ (p3FW2 (SEQ ID NO:17); 19-mer) wassynthesized.

These primers (10 pmole each) were RI-labeled at the 5′-terminus byphosphorylation with the use of [γ-³²P]ATP according to themanufacturer's instructions attached to MEGARABEL kit (manufactured byTakara Shuzo Co., Ltd.). 0.1 pmole (0.3×10⁶ cpm) of these labeledprimers and 20 μg of the total RNA of pistil or leaf were annealed inthe presence of 3 U/μl of RNase inhibitor (RNAguard, manufactured byPharmacia) in a buffer (0.25 M KCl, 2 mM Tris-HCl pH 8.0, 0.2 mM EDTA)in a reaction system of 10 μl at 40° C. for 2 hours. After adding 30 μlof another buffer (66 mM Tris-HCl pH 8.3, 6.6 mM MgCl₂, 1.3 mM DTT, 0.66mM dNTP, 130 μg/ml actinomycin D) and 1 μl (200 units) of a reversetranscriptase (M-MuLV, manufactured by BRL), the mixture was incubatedat 37° C. for 1 hour. Then ethanol and ammonium acetate were added toallow precipitation to occur. After washing the precipitate with 70%ethanol, the product was air-dried and then dissolved in anelectrophoresis buffer which was prepared by mixing the reactiontermination solution of T7 Sequencing Kit (manufactured by Pharmacia)with 0.1 M NaOH and 1 mM EDTA (2:1). A ⅓ portion of this solution washeated at 95° C. for 3 minutes and then electrophoresed on a 6%acrylamide gel. By using the same primers, an extension reaction wascarried out with T7 Sequencing Kit by using a plasmid containing RPG102as a template, and the product thus obtained was electrophoresedsimulataneously.

The results are shown in FIG. 4. No extension product was obtained fromleaf RNA in which the gene was not expressed, while 2 bands (in the caseof the C5FW primer) and 3 bands (in the case of the P3FW2 primer) ofextension products were detected by using the total RNA of pistil as thetemplate. Comparison of the sequence ladders generated side by sideindicated that the products by the two primers were detected at the sameposition in the sequence of RPG 102.

These results indicated that the transcription of RPG102 was initiatedfrom A (adenine) at 3 positions existing at intervals of several bases.A TATA box-like sequence 5′-TATATAA-3′ was found 30 bp upstream of thefirst transcription initiation point (adenine) from the 5′-terminus. Thelocation of the TATA box-like sequence coincided with genes of otherplant (Joshi, Nucleic Acids Res., 15, 6643-6653, 1987). Moreover, asdescribed above, the two ATG translation initiation codons 36 bp apartfrom each other in the same reading frame were located 77 bp and 113 bpdownstream of this transcription initiation point (FIG. 5).

(5) Determination of Whole Nucleotide Sequence of RPG102

Among the fragments formed by digesting RPG102 with PstI in Example 2(3), the fragments of 1.2, 0.8 and 0.45 kb were subcloned into the samesites of pBluescript. From the PstI 1.2 kb fragment containing thepromoter sequence, among the above-mentioned fragments, deletion cloneswith stepwise deletion of 100 to 200 bp were prepared from the bothstrands (20 clones in total) by using a deletion kit kilo-sequence for(manufactured by Takara Shuzo Co., Ltd.) and the nucleotide sequence wasdetermined with the use of M13 primer (manufactured by Takara Shuzo Co.,Ltd.) with Fluorescence Sequencer (Model 373A, manufactured by AppliedBiosystems). Regarding the fragments of 0.8 kb and 0.45 kb eachcontaining the structural gene, the nucleotide sequences were determinedby using the M13 primer (manufactured by Takara Shuzo Co., Ltd.) and theinternal primers described in the above Example 1(7). Furthermore, thenucleotide sequence in the 3′ region of RPG102 per se was determined byusing the M13 primer and the internal primers. Thus, the entirenucleotide sequence of RPG102 was finally clarified.

As a result, it was found that the whole nucleotide sequence of theRPG102 clone consisted of 2,636 bp and, when compared with thenucleotide sequence of the cDNA clone RPC175, two introns (85 bp and 199bp) were contained in the region of the structural gene. The nucleotidesequences 5′GT and AG3′ at both ends were conserved in both of theseintrons. The nucleotide sequences in the regions other than theseintrons of the genomic clone RPG102 coincided completely with the cDNAclone RPC175. A poly A signal-like sequence 5′-AATAAA-3′ (Heidecker andMessing, Annu. Rev. PlantPhysiol. 37, 439-466, 1986) was located about90 bp upstream of the EcoRI site in the 3′ side and about 40 bpdownstream of the translation termination codon TAG.

The entire nucleotide sequence of RPG 102 is represented by SEQ ID NO:3wherein the sequences in the parentheses are the introns. FIG. 6 shows acomparison of the restriction maps of RPG102 and RPC175.

Example 3 Analysis of Promoter Expression Site

(1) Construction of Vectors for Analyzing Promoter Expression andTransformation of Rice

To analyze the expression of the isolated promoter in vivo, vectorshaving GUS (β-glucuronidase) reporter gene linked thereto wereconstructed in the following manner. As described above, two ATGs werecontained in the same reading frame 77 bp and 113 bp downstream of themost upstream transcription initiation point. Since it was difficult todetermine by experiment which of the ATGs was the actual translationinitiation point, vectors for analyzing the expression of promoter wereconstructed for both of these ATGs.

An SnaBI site was located 64 bp upstream of the upstream ATG (the firstATG), i.e., 13 bp downstream of the most upstream transcriptioninitiation point, while a PstI site was located 12 bp upstream of thedownstream ATG (the second ATG) (refer to SEQ ID NOS: 1 and 3). Thesesites, were useful in the construction of the vectors from the plasmidwherein the 1.2 kb PstI fragment containing the promoter region ofRPC175 had been integrated into the PstI site of pBluescript asconstructed in the step of the nucleotide sequence analysis. In the caseof the promoter for analysis of the first ATG, this plasmid was digestedwith restriction enzymes PstI and SnaBI. Thus a promoter fragment (about1.1 kb) was cut out therefrom and then the both ends were blunted withDNA Blunting Kit (manufactured by Takara Shuzo Co., Ltd.). In the caseof the promoter for the second ATG, the fragment was digested at therestriction sites HindII and XbaI on pBluescript outside the PstI siteand thus a promoter fragment (about 1.2 kb) was cut out.

The vector used in this example was the super binary vector pSB24(Komari et al. Plant J. 10, 165-174, 1996) for Agrobacterium. Thisvector contains a GUS structural gene which in turn contains the firstintron of castor bean catalase at a downstream of CaMV35S promoter sothat the expression level will be increased by the intron. This vectorwas digested with HindIII and XbaI and the 35S promoter was eliminatedtherefrom. Then the vector was blunted (for analysis of the first ATG)at its sticky ends or not (for analysis of the second ATG) and ligatedrespectively with a blunt ended PstI-SnaBI fragment (1.1 kb) or aHindIII-XbaI fragment (1.2 kb) to thereby give vectors pYOT175IG-1 andpYOT175IG-2 each having a structure of RPC175 promoter+IGUS+NOSterminator. In order to check any possibility that thetissue-specificity might be effected by the intron, an additional vectorcarrying no intron was constructed, particularly in the case of thepromoter for the second ATG. To this end, an intron-free super binaryvector pSB21 (Komari et al. Plant J. 10, 165-174, 1996) was used. Thisplasmid was digested with HindIII and XbaI and the 35S promoter waseliminated therefrom. Then it was ligated with the above-mentionedHindIIII-XbaI fragment of about 1.2 kb to thereby give a vectorpYOT175G-2 having a structure of RPC175 promoter+GUS+NOS terminator.FIG. 7 illustrates the procedures for constructing these 3 vectors forthe expression analysis. With respect to pYOT175IG-2 and pYOT175G-2among these vectors when the translation of RPC175 was initiated fromthe upstream ATG in transformed rice cells, frame shifts of −1 type and+1 type occurred respectively, and thus the GUS protein would not betranslated.

Each vector thus constructed was transferred from E. coil intoAgrobacterium tumefaciens by tri-parental mating. Then these constructswere introduced in parallel into calli developed from immature riceembryo (“Tsukinohikari”) together with hygromycin resistance gene by theaid of Agrobacterium in accordance with the method of Hiei et al. (PlantJ., 6, 271-282, 1994). The transfer of genes was confirmed by PCR, andthe transformants were grown in a greenhouse.

(2) Analysis of Promoter Expression Site by Way of HistologicalObservation of GUS

According to the method of Jefferson et al. (EMBO J., 6, 3901-3907,1987), various organs of the rice transformants were stained for GUSwith the use of X-gluc. (5-bromo-4-chloro-3-indolyl β-D-glucuronide) asthe substrate in order to histologicaly observe the cells under astereoscopic microscope and an optical microscope. As a result, GUSexpression by the RPC175 promoter was observed in pistil stigma, anther,filament and lodicule in most of the transformants in the cases of theintron-inserted constructs (i.e., pYOT175IG-1 and pYOT175IG-2). In someplants, the GUS expression was observed in palea and lemma, leaf or root(FIG. 8).

In the case of the intron-free construct (pYOT175G-2), there were aconsiderable ratio of individual plants wherein no GUS expression wasobserved in each organ examined, though the gene transfer was confirmedby PCR and Southern analysis. The expression observed in someindividuals was in the form of spot. Moreover, the organ-specificity ofexpression well coincided with the cases of the intron-insertedconstructs (FIG. 8). Thus, it was confirmed that the existence of introndid not substantially change the tissue-specificity.

The fact that GUS expression was observed in pYOT175IG-2 and pYOT175G-2strongly indicated that the second ATG was the translation initiationpoint of the RPC175 gene.

Among the transformants showing the GUS expression at least in some ofthe tissues, the ratios of the transformants showing the GUS expressionin all of pistil, anther, filament and lodicule were 81% forpYOT175IG-1, 94% for pYOT175IG-2 and 25% for pYOT175G-2. In the case ofeach construct, about 50% of these transformants showed no expression inpalea and lemma, leaf and root, thus agreeing with the results ofNorthern analysis and RT-PCR analysis. In FIG. 8, the ordinate refers tothe number of transformants showing the expression in the specifiedorgan.

In pistil, the GUS expression was observed in stigma axis and branchedsite. Namely, the GUS gene was not expressed in the cells in the hairystigma tip (FIG. 9A). On the other hand, no GUS expression was observedin ovary. In stamen, GUS expression was observed at a high frequency infilament, in addition to anther (FIG. 9B). Moreover, GUS was highlyexpressed in vascular bundle tissues in lodicule and cells therearound(FIG. 9C).

Next, the expression time-specificity at various development stages offlower organs was examined. As a result, the strongest expression wasobserved in pistil at the heading and flowering time. At the growthstage showing a distance between auricles of the last two leaves of −5to 5 cm, the pistil of the transformants harboring the constructcarrying the inserted intron showed GUS expression. In contrast, thetransformants with the intron-free construct showed no GUS expression.These results suggest that the expression of the RPC175 promoter isstronger in the pistil at the flowering time than in the pistil at thetime of the differentiation of the hairy tissues in the top of stigma.

Although it remains unknown why the expression was observed in leaf,root or palea and lemma, the position effect of the sites of ricegenomes into which T-DNA was integrated or rearrangement of theintroduced genes may be accountable for to these results.

(3) Measurement of Promoter Activity by GUS Fluorescent Assay

From each of pYOT175IG-1 and pYOT175G-2, one line was selected so thatthe promoter expression sites as determined by the histologicalobservation of GUS in the transformation generation would coincide wellwith the results of Northern analysis and RT-PCR. Then, the GUSexpression in the next generation (R1 generation) was examined by thefluorescent analysis method with the use of MUG (4-methylumbelliferylβ-D-glucuronide) as the substrate. Leaf, root, pistil, anther(+filament), lodicule and palea and lemma were collected from one plantof the non-transformant, one R1 plant of the pYOT175IG-1 line and fourR1 plants of the pYOT175G-2 line. Then protein was extracted from eachplant and GUS was assayed. The results are shown in FIG. 10. The GUSactivities in the leaf and root of the transformants were comparable tothose of the non-transformant (18 to 210 units), while the GUSactivities in pistil, anther and lodicule of the transformants wereabout 10 to 1,000 times as high as those of the non-transformant, thoughthe activities varied from plant to plant. Namely, extremely highactivities of 486 to 38,829 units, 1,044 to 14,496 units and 1,808 to203,190 units per mg protein were observed respectively in pistil,lodicule and anther. [1 unit herein referrs to the activity by which 1pmole of 4-MU (4-methylumbelliferone) is produce from MUG in 1 minute.]Also, in palea and lemma, activities (64 to 650 units) 1 to 10 times ashigh as that of the non-transformant were observed. These factsindicated that the flower organ-specific expression of the 175 promoterwas stably maintained in the decendants of transfarmants.

Thus, it was confirmed by analyzing GUS in the generation of thetransformation and the next generation that this promoter is oneexpressed specifically in flower organs.

Example 4 Assay for Chitinase Activity of 175 Protein

(1) Expression of the Protein Encoded by RPC175 in E. coli

To examine whether the chitinase-like protein encoded by RPC175 wouldactually have chitinase activity or not, the 175 protein was firstexpressed in E. coli by using The QIA expressionist System (manufacturedby QIAGEN).

i) Construction of Expression Vector

As an expression vector pQE30 was employed. In this vector, 6 histidineresidues are positioned upstream of the multicloning site. Thus, aprotein will be expressed, by using this vector, as a fused proteinhaving a histidine tag at the N-terminus.

FIG. 11 shows the procedure for the construction. RPC175 encodes achitinase-like protein. By comparison with structures of otherchitinases it was considered that RPC175 has a leader sequenceconsisting of 20 amino acids at the N-terminus. In the geneconstruction, this leader sequence was eliminated. First, RPC175 wasdigested with PstI to prepare a fragment of about 1 kb which containedalmost all of the regions but a part of the N-terminal region of themature protein. Separately, the following two primers were synthesized:

175mat5Bm (SEQ ID NO:18), 5′-GCGGGATCCGAGCAGTGCGGCAGGCAG-3′;

C5FW2 (SEQ ID NO:19) 5′-TTGCAGTAGTCGTCGGTGAG-3′;

and PCR was carried out to amplify the remaining part of N-terminus. Theamplified product was digested with BamHI and PstI and subcloned intopBSII. After confirming the nucleotide sequence, this plasmid wasdigested with PstI and treated with CIP. Into this plasmid theabove-mentioned PstI fragment of 1 kb was inserted to construct aplasmid containing the entire region of RPC175 mature protein. Next,this plasmid was digested with BamHI and HindIII and cloned into thevector pQE30 having been digested with the same enzymes. Afterconfirming the nucleotide sequence, the vector thus obtained (namedpQE30-175ΔN) was used in the expression in E. coli.

ii) Expression in E. coli and Purification of Protein

Competent cells of E. coli M15 were prepared and transformed by theexpression vector constructed above. Plasmids were extracted fromcolonies of transformants and the introduction of the expression vectorwas confirmed. Subsequently, the E. coli cells were cultured and inducedwith IPTG in accordance with the protocol attached to the kit. Briefly,a 1/50 aliquot of the E. coli cells suspension cultured overnight wasadded to 50 ml of a 2XYT liquid medium containing ampicillin andkanamycin. After culturing for 2.5 hours, it was confirmed that theabsorbance at 600 nm (A₆₀₀) reached about 0.5. Then 2 to 4 mM of IPTGwas added and the culture was continued for additional 4.5 hours. Inaddition, two kinds of control cultures were included, namely, one whichwas free from IPTG-induction and the other which relates to E. colitransformed with the vector (pQE30) alone. The cells of each culturewere collected by centrifugation and stored at −80° C. The extraction ofcrude proteins and the purification thereof with Ni-NTA Agarose(manufactured by QIAGEN) were each carried out in accordance with themanufacturer's protocol. The crude protein extract and the purifiedprotein were electrophoresed on 12.5 to 15% SDS polyacrylamide gelaccording to the method of Laemmli (Nature 227, 680-685, 1970) and thenstained with Coomassie brilliant blue (CBB) R250. As a result, no bandseemingly assignable to the protein encoded by RPC175 was observed inthe soluble protein fraction from any of the cultures. In contrastthereto, a band with somewhat larger in size than expected (i.e., 33 kD)was observed exclusively in the insoluble fraction of theinduction-treated culture containing pQE30-175ΔN. Thus, the proteinencoded by RPC175 was mostly insoluble. To solubilize this protein, itwas necessary to add 8 M of urea to the buffer. However, the insolubleprotein was expressed in a considerably large amount and could bepurified on Ni-NTA Agarose. FIG. 12 shows the result of electrophoresisof ½ of the whole 175 protein purified from the cells cultured on ascale of 50 ml. Subsequently, to provide samples to be used for raisingan antibody, the E. coli was cultured on a 250 ml-scale and theinsoluble 175 protein was extracted and purified under the sameconditions as those described above. Then the band of the expressedprotein was cut from the polyacrylamide gel. The polyacrylamide gel bandthus cut out was further minced into pieces with a razor, thentransferred into an Eppendof tube to be ground in a homogenizer. Afteradding 10 times volume of a buffer (20 mM Tris pH 8.0, 1% SDS), themixture was shaken at room temperature over one or two nights so as toelute the protein from the acrylamide gel. After removing the gel bycentrifugation, the supernatant was dialyzed against 80% acetoneovernight in a dialysis tube Spectra/Por1 MWCO:6-8,000 (manufactured bySpectrum Medical Industries). Next, the protein solution was recoveredfrom the dialysis tube and dried.

The protein sample was suspended in 1×SDS Sample Buffer (Maniatis et al.1982) and treated at 95° C. for 5 minutes. Next, the protein waselectrophoresed on a 15% polyacrylamide gel. In order to confirm thatthe protein recovered from the cut out gel was in fact the desired one,Western blotting was performed with the use of Ni-NTA HRP conjudgate(manufactured by QIAGEN) as the antibody. On the other hand, the gelafter the completion of the electrophoresis was stained with CBB and theprotein concentration was estimated by comparing with markers of knownconcentrations (Prestained SDS-PAGE standards Low Range, manufactured byBIORAD).

iii) Production of Antibody

The production of antibody was undertaken by Sawady Technology Co., Ltd.When determined by ELISA, the rabbit antibody had a titer of 23,600. Asa result of Western analysis with the use of HRP as the secondaryantibody, this antibody reacted with the sample protein with a highsensitivity.

iv) Solubilization of Protein Encoded by RPC175

As described above, the protein encoded by RPC175, when expressed in E.coli, was mostly (99% or above) insoluble. When the soluble fraction wassubjected to Western blotting with the use of the above-mentionedantibody, on the other hand, the 175 protein was detected though in asmall amount. Moreover, it was also found that this protein contained ina trace amount could be purified on Ni-NTA Agarose. Generally speaking,when a foreign protein is expressed in E. coli, the protein often cannotassume the correct folded structure but forms inclusion bodies due torapid induction of expression. This phenomenon can be avoided byemploying milder induction conditions. To obtain a large amount of 175protein in the soluble form, therefore, the following experiment wascarried out with respect to the IPTC concentration and culturetemperature which were the main factors of the induction conditions.Namely, the induction was performed under three conditions (at 37° C.with 2 mM of IPTG; at 25° C. with 0.5 mM of IPTG; and at 15° C. with 0.1mM of IPTG). The culture was continued for 4.5 hours at 37° C. and 25°C. and for 18 hours at 15° C. After culturing under the conditions asspecified above, the cells showed turbidities (A₆₀₀) of 1.04, 0.89 and0.85 respectively at 37° C., 25° C. and 15° C. The E. coli cells (in 50ml liquid culture medium:) which expressed the protein under theseconditions were collected by centrifugation and stored at −80° C. Fromthese cells, proteins were extracted by using a urea-free buffersolution in accordance with QIAGEN's instructions and the 175 proteincarrying the HIS tag was purified with the use of Ni-NTA Agarose.Finally, the 175 protein was eluted from the Ni-NTA Agarose with 300 μlof a 0.1 M phosphate buffer (pH 4.5) containing 10 mM of Tris. Theeluate (10 μl) was electrophoresed on SDS-PAGE followed by Westernanalysis with the use of the above-mentioned antibody against the 175protein. Since it was anticipated that the 175 protein in the sampleswas only in a trace amount, ECL+plus System (manufactured by Amersham)was used in the Western blotting. The primary and secondary antibodieswere added each at a concentration of 1/10,000 and reacted each time for1 hour with the ECL nitrocellulose membrane having the fractionatedproteins blotted thereon. The reaction with the substrate was continuedfor 5 minutes and the X-ray film was exposed to light for 2 to 20minutes. As a result, the densities of the bands assignable to thepurified soluble 175 protein increased as the IPTG concentration and theculture temperature of E. Coli were lowered as shown in FIG. 13. Thedensities of these bands were compared with that of the bands of theabove-mentioned 175 protein of a known concentration (prepared bydissolving 10 ng of the insoluble fraction prepared for the productionof the antibody in 8 M urea) electrophoresed on the same gel. As aresult, the soluble 175 protein was obtained in amounts of 97.6 ng, 12.4ng and 2.1 ng in the order of how mild the culture conditions were. Atthe same time, 10 μl aliquots of the whole proteins eluted from theNi-NTA Agarose gel were quantitated with Bio-Rad Protein Assay(manufactured by BIORAD). As a result, the protein contents wererespectively 38 μg, 24 μg and 34 μg. Thus, the ratios of the 175 proteinin the whole proteins eluted were calculated respectively as 2.57%,0.52% and 0.06%. These results suggest that the ratio of the soluble 175protein could be elevated by lowering the IPTG concentration and theculture temperature.

(2) Assay of Chitinase Activity of E. coli

Chitinase activity was assayed by the Reissig method by determining thesaccharides solubilized from colloidal chitin as the substrate. Thecolloidal chitin was prepared in the following manner. Chitin powder 2 gwas dissolved gradually in 100 ml of cold conc. hydrochloric acid whileelevating temperature and then filtered through a G-3 glass filter. Thefiltrate was added slowly to 10 times volume of sterilized water andallowed to stand at 4° C. overnight to re-precipitate the chitin. Afterremoving the supernatant, the precipitate was re-suspended in sterilizedwater and centrifuged at 6,000 g for 10 minutes. The washing wasrepeated until the pH of the supernatant became neutral. The precipitatewas finally suspended in 150 ml of sterilized water to give a colloidalchitin solution.

The chitinase activity was measured in the following manner. A 100 μlaliquot of the enzyme solution and 100 μl of the colloidal chitinsolution were mixed and incubated at 37° C. for 2 hours. Aftercentrifuging at 6,000 rpm for 5 minutes, 150 μl of the supernatant wascollected. As the blanc test same enzyme solution alone was incubated at37° C. for 2 hours and then colloidal chitin was added immediatelybefore centrifugation. To each of these supernatants, 15 μl of a 1 Mphosphate buffer (pH 7.2) was added to adjust the pH value.Subsequently, 10 μl of 3% Helicase (manufactured by SIGMA) was added andthe mixture was incubated at 37° C. for 1 hour to allow the chitinoligomers to be hydrolyzed. Next, 30 μl of 0.8 M potassium borate-KOH(pH 10.2) was added and the mixture was boiled for 3 minutes.Simultaneously, 25, 50 and 100 nmol N-acetylglucosamine (GlcNAC,manufactured by SIGMA) solubilized in the above assay reagents were alsoboiled to provide a standard curve. After the completion of boiling, themixtures were immediately ice-cooled followed by addition of 1 ml of asolution of p-dimethyl aminobenzaldehyde (DMAB, manufactured by WakoPure Chemical Industries, Inc.) prepared by dissolving 1 g of DMAB in100 ml of acetic acid containing 1% of hydrochloric acid. Afterincubating at 37° C. for 20 minutes, the absorbance (A₅₈₅) was measuredand the amount of GlcNAc was calculated from the standard curve. Oneunit is defined as the activity of the enzyme which cause solubilizationof saccharides corresponding to 1 μmol of N-acetylglucosamine in 1minute.

By this assay system, there were measured the chitinase activities ofthe purified 175 protein carrying the HIS tag produced by E. coli cellscultured under the above-mentioned three expression-inducing conditions.Further, the proteins in the two kinds of control cultures [i.e., onehaving E. Coli with the vector (pQE30) alone and the other being freefrom IPTG-induction] were extracted, purified and subjected to theassay. As a result, an apparent chitinase activity was detected in thetest wherein pQE30-175ΔN was subjected to the induction of expression atthe culture temperature of 15° C. and IPTG concentration of 0.1 mM. Theenzyme activity in this culture was 1.9 mU/mg protein, i.e., 3 to 4times as high as those in the control cultures (0 to 5.1 mU/mg protein).However, it is to be understood that this activity was based on thewhole proteins eluted from the Ni-NTA Agarose. As described above, the175 protein amounted to about 2.57% of the eluted proteins. Thus, it isestimated that the enzyme activity of the 175 protein is at leastseveral ten mU/mg protein. In the test lot of the culture temperature of25° C., a slight chitinase activity, compared with the control lots, wasdetected. However, the test lot of the culture temperature of 37° C.showed no activity. This is seemingly because the 175 protein subjectedto the assay had only a low concentration.

Based on these results, it has been clarified that the chitinase-likeprotein encoded by RPC175 has actually a chitinase activity.

TABLE 2 Chitinase activity of protein encoded by RPC175 gene Culturetemp. IPTC concn. Activity (° C.) Expression vector (mM) (mU/mg protein)15 pQE30 0   0   15 pQE30 0.1 0.50 15 pQE30-175ΔN 0   0.51 15pQE30-175ΔN 0.1 1.90 25 pQE30 0   0.99 25 pQE30 0.5 1.04 25 pQE30-175ΔN0   1.48 25 pQE30-175ΔN 0.5 1.62 37 pQE30 0   0.35 37 pQE30 2   0.72 37pQE30-175ΔN 0   0.62 37 pQE30-175ΔN 2   0.68

EFFECTS OF THE INVENTION

According to the present invention, it becomes possible to geneticallymanipulate flower organs not only anther but also pistil or lodicule ofplants. Thus female sterile plants and rice plants with exposed stigmamay be constructed. Also, the flowering characteristics may bephysiologically regulated. The present invention further makes itpossible to construct plants which are resistant against pathogenicbacteria and fungi containing chitin.

19 1 1318 DNA Oryza sativa CDS (114)..(1097) Clone RPC175; Library ZAPIIcDNA library from pistil mRNA; Strain IR24; 1 atcactcacc agctacgtacactcaaccaa cacaccactg aaaagcaaga ttttgttgaa 60 gaaataagca tcttgccatgatcgcagcaa gggctgcaaa cctgcaggtg gcc atg 116 Met 1 aag gcc ctg gcg ctggcc gtg ctg gcc ctc gcc tac gcc gcg gcg acg 164 Lys Ala Leu Ala Leu AlaVal Leu Ala Leu Ala Tyr Ala Ala Ala Thr 5 10 15 gcg cgc gcc gag cag tgcggc agg cag gcc ggc ggc gcc agg tgc ccc 212 Ala Arg Ala Glu Gln Cys GlyArg Gln Ala Gly Gly Ala Arg Cys Pro 20 25 30 aac agg ctc tgc tgc agc aggtgg ggg tgg tgc ggc ctc acc gac gac 260 Asn Arg Leu Cys Cys Ser Arg TrpGly Trp Cys Gly Leu Thr Asp Asp 35 40 45 tac tgc aag ggc ggc tgc cag agccag tgc cgc gtc tcc cgc gac ggc 308 Tyr Cys Lys Gly Gly Cys Gln Ser GlnCys Arg Val Ser Arg Asp Gly 50 55 60 65 ggc gac gac gac gtc gcc gcg gtgctg ctc acg gcg ccg ggc ggc ggc 356 Gly Asp Asp Asp Val Ala Ala Val LeuLeu Thr Ala Pro Gly Gly Gly 70 75 80 cgc gcc ggc gtg gcg tcc gtc gtg acgtcg gac cag ttc gag cgc atg 404 Arg Ala Gly Val Ala Ser Val Val Thr SerAsp Gln Phe Glu Arg Met 85 90 95 ctg ccc cac cgc gac gac gcg gcg tgc cccgcc cgc ggg ttc tac gcc 452 Leu Pro His Arg Asp Asp Ala Ala Cys Pro AlaArg Gly Phe Tyr Ala 100 105 110 tac cgc gcc ttc gtc gcc gcg gcc ggc gcgttc ccg gcc ttc gcc gcc 500 Tyr Arg Ala Phe Val Ala Ala Ala Gly Ala PhePro Ala Phe Ala Ala 115 120 125 acg ggc gac gcc gac acc cgc aag cgt gaggtc gcc gcg ttc ctg gcc 548 Thr Gly Asp Ala Asp Thr Arg Lys Arg Glu ValAla Ala Phe Leu Ala 130 135 140 145 cag act tcc cac gcg acc tct ggt gggccc tac tcg tgg ggc tac tgc 596 Gln Thr Ser His Ala Thr Ser Gly Gly ProTyr Ser Trp Gly Tyr Cys 150 155 160 tac aag gag gtg aag ggc gcg acg tcagac ttc tgc gtg ccg aac gcg 644 Tyr Lys Glu Val Lys Gly Ala Thr Ser AspPhe Cys Val Pro Asn Ala 165 170 175 cgc tgg ccg tgc gcg ccc ggc aag gcgtac cac gcc cgc gga ccc atg 692 Arg Trp Pro Cys Ala Pro Gly Lys Ala TyrHis Ala Arg Gly Pro Met 180 185 190 caa atc gca tac aac tac aac tat ggggcg gcc ggc gag gcg atc ggc 740 Gln Ile Ala Tyr Asn Tyr Asn Tyr Gly AlaAla Gly Glu Ala Ile Gly 195 200 205 gcg gac ctg ctg ggc aac ccg gag ctggtg gca acg gac ccg acg gtg 788 Ala Asp Leu Leu Gly Asn Pro Glu Leu ValAla Thr Asp Pro Thr Val 210 215 220 225 gcg ttc aag acg gcg ctg tgg ctgtgg atg acc gcg cgg tcg ccg agc 836 Ala Phe Lys Thr Ala Leu Trp Leu TrpMet Thr Ala Arg Ser Pro Ser 230 235 240 cag ccg tcg ccg cac gcc gtc gtcacg ggg cag tgg act ccg act ccc 884 Gln Pro Ser Pro His Ala Val Val ThrGly Gln Trp Thr Pro Thr Pro 245 250 255 gcg gac agc gcg gcc ggc cgc gcgcca ggc tac ggg ctc acc acg aac 932 Ala Asp Ser Ala Ala Gly Arg Ala ProGly Tyr Gly Leu Thr Thr Asn 260 265 270 atc ctc acc ggc ggg ctc cag tgcgcc ggc ggc aac ggc ggc gcc gac 980 Ile Leu Thr Gly Gly Leu Gln Cys AlaGly Gly Asn Gly Gly Ala Asp 275 280 285 cgg gtc gcg ttc tac aag cgc tactgc gac gtg ctc ggc gtc ggc tac 1028 Arg Val Ala Phe Tyr Lys Arg Tyr CysAsp Val Leu Gly Val Gly Tyr 290 295 300 305 ggg ccc aac ctg gac tgc ttcggc cag gcg ccg ttc gac ggc gac atc 1076 Gly Pro Asn Leu Asp Cys Phe GlyGln Ala Pro Phe Asp Gly Asp Ile 310 315 320 atg tcg gcg tct gcg gcg aagtagacgtgtg cgccgccgtg ccggccccga 1127 Met Ser Ala Ser Ala Ala Lys 325tcgatcgaat aaaattgcgt gtgagtacgc acttcgcacg gtcgctctgc agccagagtg 1187agtgagtttg ctttatgtat ttttcggttt cgggcgagga attcttcatg gatctgtgaa 1247agcccatatg tatgcatggt catggcatga ataaagtagt actgatcttc tcgaaaaaaa 1307aaaaaaaaaa a 1318 2 328 PRT Oryza sativa Clone RPC175; Library ZAPIIcDNA library from pistil mRNA; Strain IR24; 2 Met Lys Ala Leu Ala LeuAla Val Leu Ala Leu Ala Tyr Ala Ala Ala 1 5 10 15 Thr Ala Arg Ala GluGln Cys Gly Arg Gln Ala Gly Gly Ala Arg Cys 20 25 30 Pro Asn Arg Leu CysCys Ser Arg Trp Gly Trp Cys Gly Leu Thr Asp 35 40 45 Asp Tyr Cys Lys GlyGly Cys Gln Ser Gln Cys Arg Val Ser Arg Asp 50 55 60 Gly Gly Asp Asp AspVal Ala Ala Val Leu Leu Thr Ala Pro Gly Gly 65 70 75 80 Gly Arg Ala GlyVal Ala Ser Val Val Thr Ser Asp Gln Phe Glu Arg 85 90 95 Met Leu Pro HisArg Asp Asp Ala Ala Cys Pro Ala Arg Gly Phe Tyr 100 105 110 Ala Tyr ArgAla Phe Val Ala Ala Ala Gly Ala Phe Pro Ala Phe Ala 115 120 125 Ala ThrGly Asp Ala Asp Thr Arg Lys Arg Glu Val Ala Ala Phe Leu 130 135 140 AlaGln Thr Ser His Ala Thr Ser Gly Gly Pro Tyr Ser Trp Gly Tyr 145 150 155160 Cys Tyr Lys Glu Val Lys Gly Ala Thr Ser Asp Phe Cys Val Pro Asn 165170 175 Ala Arg Trp Pro Cys Ala Pro Gly Lys Ala Tyr His Ala Arg Gly Pro180 185 190 Met Gln Ile Ala Tyr Asn Tyr Asn Tyr Gly Ala Ala Gly Glu AlaIle 195 200 205 Gly Ala Asp Leu Leu Gly Asn Pro Glu Leu Val Ala Thr AspPro Thr 210 215 220 Val Ala Phe Lys Thr Ala Leu Trp Leu Trp Met Thr AlaArg Ser Pro 225 230 235 240 Ser Gln Pro Ser Pro His Ala Val Val Thr GlyGln Trp Thr Pro Thr 245 250 255 Pro Ala Asp Ser Ala Ala Gly Arg Ala ProGly Tyr Gly Leu Thr Thr 260 265 270 Asn Ile Leu Thr Gly Gly Leu Gln CysAla Gly Gly Asn Gly Gly Ala 275 280 285 Asp Arg Val Ala Phe Tyr Lys ArgTyr Cys Asp Val Leu Gly Val Gly 290 295 300 Tyr Gly Pro Asn Leu Asp CysPhe Gly Gln Ala Pro Phe Asp Gly Asp 305 310 315 320 Ile Met Ser Ala SerAla Ala Lys 325 3 2636 DNA Oryza sativa CDS (1235)..(1691) CDS(1777)..(1909) CDS (2109)..(2502) Clone RPG102; Library dashII genomiclibrary from green leaf genome DNA; Strain IR24 3 4 328 PRT Oryza sativaClone RPG102; Library dashII genomic library from green leaf genome DNA;Strain IR24 4 Met Lys Ala Leu Ala Leu Ala Val Leu Ala Leu Ala Tyr AlaAla Ala 1 5 10 15 Thr Ala Arg Ala Glu Gln Cys Gly Arg Gln Ala Gly GlyAla Arg Cys 20 25 30 Pro Asn Arg Leu Cys Cys Ser Arg Trp Gly Trp Cys GlyLeu Thr Asp 35 40 45 Asp Tyr Cys Lys Gly Gly Cys Gln Ser Gln Cys Arg ValSer Arg Asp 50 55 60 Gly Gly Asp Asp Asp Val Ala Ala Val Leu Leu Thr AlaPro Gly Gly 65 70 75 80 Gly Arg Ala Gly Val Ala Ser Val Val Thr Ser AspGln Phe Glu Arg 85 90 95 Met Leu Pro His Arg Asp Asp Ala Ala Cys Pro AlaArg Gly Phe Tyr 100 105 110 Ala Tyr Arg Ala Phe Val Ala Ala Ala Gly AlaPhe Pro Ala Phe Ala 115 120 125 Ala Thr Gly Asp Ala Asp Thr Arg Lys ArgGlu Val Ala Ala Phe Leu 130 135 140 Ala Gln Thr Ser His Ala Thr Ser GlyGly Pro Tyr Ser Trp Gly Tyr 145 150 155 160 Cys Tyr Lys Glu Val Lys GlyAla Thr Ser Asp Phe Cys Val Pro Asn 165 170 175 Ala Arg Trp Pro Cys AlaPro Gly Lys Ala Tyr His Ala Arg Gly Pro 180 185 190 Met Gln Ile Ala TyrAsn Tyr Asn Tyr Gly Ala Ala Gly Glu Ala Ile 195 200 205 Gly Ala Asp LeuLeu Gly Asn Pro Glu Leu Val Ala Thr Asp Pro Thr 210 215 220 Val Ala PheLys Thr Ala Leu Trp Leu Trp Met Thr Ala Arg Ser Pro 225 230 235 240 SerGln Pro Ser Pro His Ala Val Val Thr Gly Gln Trp Thr Pro Thr 245 250 255Pro Ala Asp Ser Ala Ala Gly Arg Ala Pro Gly Tyr Gly Leu Thr Thr 260 265270 Asn Ile Leu Thr Gly Gly Leu Gln Cys Ala Gly Gly Asn Gly Gly Ala 275280 285 Asp Arg Val Ala Phe Tyr Lys Arg Tyr Cys Asp Val Leu Gly Val Gly290 295 300 Tyr Gly Pro Asn Leu Asp Cys Phe Gly Gln Ala Pro Phe Asp GlyAsp 305 310 315 320 Ile Met Ser Ala Ser Ala Ala Lys 325 5 25 DNA Oryzasativa RPC15 5 gtcagacacn nagtagtgag tggtc 25 6 12 DNA Oryza sativaRPC17 6 tcatcactca cc 12 7 29 DNA Artificial Sequence Description ofArtificial Sequenceplasmid psB24 7 tacctagaac atggatccct acagcgtaa 29 835 DNA Artificial Sequence Description of Artificial SequenceplasmidpsB24 8 ctgcaccccg ggggatccac tagttctaga acatg 35 9 61 DNA ArtificialSequence Description of Artificial Sequenceplasmid psB21 9 ctgcagcccgggggatccac tagttctaga ggatcccccg ggtggtcagt cccttatgtt 60 a 61 10 17 DNAArtificial Sequence Description of Artificial Sequencesynthetic primer75RV1 10 gacacccgca agcgtga 17 11 17 DNA Artificial Sequence Descriptionof Artificial Sequencesynthetic primer 75FW1 11 cccttcacct ccttgta 17 1224 DNA Artificial Sequence Description of Artificial Sequencesyntheticprimer 12 gtatccatga gactacatac aact 24 13 22 DNA Artificial SequenceDescription of Artificial Sequencesynthetic primer 13 tactcagccttggcaatcca ca 22 14 21 DNA Artificial Sequence Description of ArtificialSequencesynthetic primer 175RV1 14 gacatcatgt cggcgtctgc g 21 15 23 DNAArtificial Sequence Description of Artificial Sequencesynthetic primer175FW1 15 gccatgacca tgcatacata tgg 23 16 24 DNA Artificial SequenceDescription of Artificial Sequencesynthetic primer C5FW 16 cttcatggccacctgcaggt ttgc 24 17 19 DNA Artificial Sequence Description ofArtificial Sequencesynthetic primer p3FW2 17 tgcgatcatg gcaagatgc 19 1827 DNA Artificial Sequence Description of Artificial Sequencesyntheticprimer 175mat5Bm 18 gcgggatccg agcagtgcgg caggcag 27 19 20 DNAArtificial Sequence Description of Artificial Sequencesynthetic primerC5FW2 19 ttgcagtagt cgtcggtgag 20

What is claimed is:
 1. An isolated DNA fragment comprising the sequencefrom position 1 to 1234 in the nucleotide sequence of SEQ ID NO: 3, or apart of said sequence having a promoter activity specific to a floralorgan.
 2. An isolated DNA fragment as claimed in claim 1, wherein saidpart of the sequence from positions 1 to 1234 in the nucleotide sequenceof SEQ ID NO:3 is a sequence from the upstream region of thetranscription initiation point.
 3. An isolated DNA fragment as claimedin claim 1, wherein said part comprises a sequence of 500 contiguousnucleotides which precedes the transcription initiation point.
 4. Anisolated DNA fragment comprising the sequence from position 1 to 1140 inthe nucleotide sequence of SEQ ID NO: 3, or a part of said sequencehaving a promoter activity specific to a floral organ.
 5. An isolatedDNA fragment comprising the sequence from position 1 to 1121 in thenucleotide sequence of SEQ ID NO: 3, or a part of said sequence having apromoter activity specific to a floral organ.
 6. An isolated DNAfragment comprising a nucleotide sequence which is hybridizable underthe following conditions of hybridization and washing: hybridization:0.25 M Na₂HPO₄, pH 7.2, 7% SDS, 1 mM EDTA, 1×Denhardt's solution, 68°C., overnight; washing: 20 mM Na₂HPO₄, pH 7.2, 1% SDS, 1 mM EDTA, 68°C., 15 min. twice. with a DNA sequence comprising the sequence frompositions 1 to 1234 in the nucleotide sequence of SEQ ID NO: 3, whereinsaid hybidizable sequence has a promoter activity specific to a floralorgan.
 7. The isolated DNA fragment of claim 6, wherein said DNAfragment is ligated to a gene, and said gene is a structural gene thatcodes for a protein capable of inducing the sterility in plants.
 8. Theisolated DNA fragment of claim 6, wherein said DNA fragment is ligatedto a gene, and said gene codes for a protein that is capable ofpromoting the elongation or division of plant cells.
 9. The isolated DNAfragment of claim 6, wherein said DNA fragment is ligated to a gene, andsaid gene codes for a protein that improves tolerance to herbicides ordiseases.
 10. An isolated DNA fragment comprising a nucleotide sequencewhich is hybridizable under the following conditions of hybridizationand washing: hybridization: 5×SSC, 5×Denhardt's solution, 1% SDS, 68°C., overnight; washing: 0.2×SSC, 0.1% SDS, 42° C., 15 min. twice; with aDNA sequence comprising the sequence from positions 1 to 1234 in thenucleotide sequence of SEQ ID NO: 3, wherein said hybridizable sequencehas a flower organ-specific promoter activity.
 11. The isolated DNAfragment as claimed in claim 6 which comprises positions 300-1234nucleotides of SEQ ID NO:3.